Cloning, Expression and Characterization of Recombinant Human Fc Receptor Like 1, 2 and 4 Molecules

Authors

Azam Hemmati Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, IR Iran

Fazel Shokri Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, IR Iran

Hodjatallah Rabbani Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, IR Iran

Jalal Khoshnoodi Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, IR Iran

Mahdi Shabani Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, IR Iran and Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, IR Iran

Mahdi Zandemami Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, IR Iran

Mahmood Jeddi-Tehrani Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, IR Iran

Zahra Amirghofran Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, IR Iran

Abstract:

Background: The Fc receptor like (FCRL) molecules belong to the immunoglobulin (Ig) superfamily with potentially immunoregulatory function. Among the FCRL family FCRL2 and 4 are predominantly expressed on memory B cells and FCRL1 is a pan- B cell marker. To date, no ligand has been identifid for the human FCRL1, 2 and 4 molecules. Objectives: Cloning, expression, purifiation and structural analysis of the extracellular domain of human FCRL1, 2 and 4 proteins. Materials and Methods: In this study, the extracellular part of human FCRL1, 2 and 4 were subcloned into prokaryotic expression vectors pET-28b (+) and transformed into BL21-DE3 E.coli strain. Protein expression was optimized by fie adjustments such as induction time, incubation temperature and expression hosts. Recombinant FCRL proteins were purifid by metal affity chromatography using Ni-NTA resin. Purifid FCRL proteins were further characterized by SDS-PAGE and immunoblotting using His-tag and FCRL specifi polyclonal antibodies. Results: Our results demonstrated that FCRL1, 2 and 4 were successfully expressed in pET-28b (+) vector. Optimization of the expression procedure showed that IPTG induction at OD600 = 0.9 and overnight incubation at 37˚C resulted in the highest expression levels of FCRL proteins ranging from approximately 15% (FCRL1) to 25% (FCRL2 and 4) of the total bacterial lysate proteins. Conclusions: These purifid recombinant proteins are potentially a valuable tool for investigating the immunoregulatory function of FCRL molecules and the production of specifi mAbs for immunotherapeutic interventions.

Upgrade to premium to download articles

Already have an account?login

similar resources

cloning, expression and characterization of recombinant human fc receptor like 1, 2 and 4 molecules

background: the fc receptor like (fcrl) molecules belong to the immunoglobulin (ig) superfamily with potentially immunoregulatoryfunction. among the fcrl family fcrl2 and 4 are predominantly expressed on memory b cells and fcrl1 is a pan- b cell marker. to date, noligand has been identifid for the human fcrl1, 2 and 4 molecules.objectives: cloning, expression, purifiation and structural analysi...

full text

Cloning, Expression, Purification and CD Analysis of Recombinant Human Betatrophin

Betatrophin is a member of the angiopoietin-like (ANGPTL) family that has been implicated in both triglyceride and glucose metabolism. The physiological functions and molecular targets of this protein remain largely unknown; hence, a purified available protein would aid study of the exact role of betatrophin in lipid or glucose metabolism. In this study, we cloned the full-length cDNA of betatr...

full text

Toll Like Receptor 2 and 4 Expression in Peripheral Blood Mononuclear Cells of Multiple Sclerosis Patients

Background: Multiple sclerosis (MS) is a T cell mediated autoimmune disease with unknown etiology. Appropriate MS therapeutic strategies need thorough understanding of both disease etiology and pathogenesis mechanisms. Ligation of TLR-2 and TLR-4 stimulates the production of several cytokines leading to CNS autoimmunity and neurodegenerative diseases. Objective: To find a relationship between M...

full text

Cloning and expression of tetanus toxin C fragment (Fc) in prokaryotic vector for constructing recombinant protein based vaccine for tetanus

Tetanus is a disease caused by tetanus toxin, a potent inhibitor for the release of inhibitory neurotransmitter in the central nervous system that causes spastic paralysis. Fragment C (52 kD) of this toxin is responsible for binding to the neuronal membrane. For this reason, and also its non toxigenic and immunogenic nature, this fragment might be ideal for new vaccine development. Presently, w...

full text